Impact of Preharvest Relative Air Humidity and Postharvest Modified Atmosphere Packaging on Cucumber Fruit Quality
By
Haider Ali
Master’s Thesis 2017 (60 Credits)
Master of Science in Plant Sciences
Department of Plant Sciences Faculty of Biosciences
Norwegian University Of Life Sciences, Norway
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The Norwegian University of Life Sciences Norges Miljø og Biovitenskapelige Universitet
Master Thesis
Impact of Preharvest Relative Air Humidity and Postharvest Modified Atmosphere Packaging on Cucumber Fruit Quality
By
Haider Ali
Department of Plant Sciences Institutt for Plantevitenskape
Norwegian University Of Life Sciences, Norway P.O Box 5003
1432, Ås, Norway
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Acknowledgements
It is quite delectable to become and to avail this most propitious opportunity to articulate with utmost gratification, my profound and intense sense of indebtedness to my ever affectionate supervisor, Dr. Sissel Torre, Professor, Department of Plant Sciences, Faculty of Biosciences, Norwegian University of Life Sciences, Norway. Faisalabad. Her proficient counseling, valuable suggestions, boundless forbearance, indefatigable help with anything, anywhere, anytime, consummate advice and thought-provoking instructions in piloting this research venture and to reach its present effective culmination. Special thanks for her would always be due.
I am unfathomable indebted to Professor Knut Asbjørn Solhaug for his keen interest, dedication and guidance and valuable suggestions during research work. I feel my words so shallow; they do not seem to be the same as felt to be thankful to my worthy research coordinator, Ida Kristin Hagen for her help in setting up my experiment and making sure that everything ran smoothly.
I do not have words at command in acknowledging that all credit goes to my affectionate guardian and my brother, Muhammad Sohail Mazhar and my loving Mother for their amicable attitude and love, immense orison, mellifluous affections, inspiration, well-wishing and keen interest which hearten me to achieve success in every sphere of life. Their prayers are the roots of my success. I cannot ignore my brother and sister, who has always inspired and encouraged me and their prayers have been with me and will always be with me for my success.
I am very thankful to Per Osmund Espedal, the cucumber fruit grower, who provided me the cucumber fruits for my research experiment. Furthermore, I am really grateful to StePac L.A.
Ltd. Tefen, Israel for their Modified atmosphere packaging bags, which were used in my research experiment.
Haider Ali
iv Table of Contents
Abstract ... 1
1. Introduction ... 3
1.1 Objective of study ... 4
1.2 Literature review ... 5
1.2.1Cucumber ... 5
1.2.2 Cucumber industry in the world ... 5
1.2.3 Cucumber industry in Norway ... 6
1.2.4 Quality ... 6
1.2.5 Nutritional importance ... 6
1.2.6 Therapeutic importance ... 7
1.2.7 Climate and plant growth ... 7
1.2.8 Relative Air Humidity and Plant Morphology ... 8
1.2.9 Carbohydrates and polyphenols in cucumber ... 9
1.2.10 Greenhouse relative humidity and Fruit quality ... 9
1.2.11 MAP bags and fruit physiology ... 10
1.2.12 MAP bags and chilling injury ... 10
1.2.13 Exogenous ABA application and fruit quality ... 11
2. Materials and Methods ... 12
2.1 Experiment 1: effect of relative air humidity on plant growth and fruit quality ... 12
2.1.1 Seedling Production ... 12
2.1.2 Experiment set-up ... 13
2.1.3 Irrigation and plant maintenance ... 13
2.1.4 Data collection ... 14
2.1.5 Growth data and Physical Analysis ... 14
2.1.6 Biochemical analysis ... 15
2.1.6.1 Total Phenolic and Anti-oxidants capacity (Fruits) ... 15
2.1.6.1.1 Anti-Oxidant activity (FRAP- assay) ... 15
2.1.6.1.2 Total phenolic contents (TPC) determination... 16
2.1.6.2 Polyphenols compounds ... 16
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2.1.6.3 Carbohydrates ... 20
2.1.6.4 Mineral Analysis ... 21
2.1.7 Organoleptic Evaluation ... 22
2.2 Experiment 2: storability of commercial fruits ... 23
2.2.1 Experimental layout ... 23
2.2.2 Data collection ... 24
2.2.2.1 Respiration ... 24
2.2.2.2 Physical data ... 24
2.2.2.2.1 Weight Loss ... 24
2.2.2.2.2 Skin shriveling and disease incidence ... 24
2.2.2.3 Ion Leakage ... 24
2.2.2.4Total Phenolic and Anti-oxidants (Fruits) ... 24
3. Results ... 25
3.1 Experiment 1: effect of relative air humidity on plant growth and fruit quality ... 25
3.1.1 Comparison of growth and morphology of cucumber plants and fruits produced in different RH conditions ... 25
3.1.1.1 Growth and Morphological parameters ... 25
3.1.1.1.1 15 days under controlled conditions ... 25
3.1.1.1.2 On harvest ... 25
3.1.2 Biochemical analysis of cucumber leaves and fruits under different relative air humidity conditions ... 27
3.1.2.1 Biochemical analysis ... 27
3.1.2.1.1 Total phenolics and Anti-oxidant capacity (FRAP) ... 27
3.1.2.1.2 Polyphenols in cucumber plant leaves, fruit pulp and peel ... 27
3.1.2.1.3 Carbohydrates in cucumber plant leaves and fruits ... 35
3.1.2.1.3.1 Carbohydrates in Leaves... 37
3.1.2.1.3.2 Carbohydrates in Fruits ... 38
3.1.3 Comparison of mineral contents in cucumber leaves and fruits from various RH conditions 39 3.1.3.1 Mineral contents in fruit ... 39
3.1.3.2 Mineral contents in leaves ... 40
3.1.4 Sensory analysis of cucumber fruits from various RH conditions ... 42
3.1.4.1 Determining the key attributes ... 42
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3.1.4.2 Difference between Sensory perception of cucumber fruits from different RH
conditions. ... 43
3.1.4.3 Correlation analysis between Sensory attributes, sugars and minerals contents in cucumber fruits sample ... 45
3.2 Experiment 2 ... 47
3.2.1 Respiration of cucumber fruits from different treatments and different storage period under temperature condition ... 47
3.2.2 Physical weight loss (percentage) in cucumber fruits from different treatments at different removals kept at different stages (various temperature conditions) ... 48
3.2.3 Disease incidence (%) in cucumber fruits from different treatments at different removals kept at different stages (various temperature conditions) ... 51
3.2.4 Skin shriveling in cucumber fruits from different treatments at different removals and different at different stages (various temperature conditions) ... 51
3.2.5 Ion leakage (percentage) in cucumber fruits from different treatments at different removals and different at different stages (various temperature conditions) ... 53
3.2.6 Comparison of anti-oxidants capacity (FRAP) and total phenolics in cucumber fruits from different treatments at different removals ... 56
4. Discussion ... 57
4.1 Experiment 1 ... 57
4.1.1 Plant growth and morphology ... 57
4.1.2 Fruit growth and morphology ... 58
4.1.3 Antioxidant capacity, total phenolics contents and polyphenols concentration ... 58
4.1.4 Carbohydrates concentration ... 58
4.1.5 Nutrients concentration ... 59
4.1.6 Sensory analysis of fruits from different relative air humidity conditions ... 60
4.2 Experiment 2 ... 61
4.2.1 MAP bags influence on cucumber fruit respiration and physical weight loss ... 61
4.2.2 MAP bags influence on disease incidence and chilling injury in cucumber fruits ... 61
Conclusion ... 62
References ... 63
Appendix ... 71
1
Abstract
The effect of relative air humidity (RH) was tested on the cucumber (Cocktail, Quarto F1 cultivar) plant growth, morphology and fruit quality in controlled growth chambers. The plants were grown at moderate RH (60%) and high RH (90%) with the same temperature, CO2 and irradiance. In addition, another experiment was conducted to test that effect of exogenous application of abscisic acid (ABA) and different packaging materials (modified atmosphere packaging bags and plastic folio) on the quality of commercial produced cucumber fruits stored at low temperature (TC) storage for 14 days and 21 days.
Cucumber plants and fruits responded strongly to the different RH conditions. The plant shoot length, number of leaves and fruit diameter was increased at high RH, while average leaf area, relative chlorophyll contents, number of side shoots were increased at moderate RH compared to high RH. Higher antioxidant capacity and total phenolics contents were observed in fruits from moderate RH. Through High Pressure Liquid Chromatography (HPLC) analysis 3 polyphenols (resveratrol, luteolin and apigenin) were identified in cucumber leaf samples and 6 polyphenols (apigenin, luteolin, quercetin 3 glycoside, quercetin, pinoresinol and resveratrol) were found in cucumber fruit samples. Moderate RH increased the resveratrol and luteolin concentration in cucumber leaves and increased the luteolin, quercetin 3 glycoside, quercetin and pinoresinol in cucumber fruit sample. Moderate RH not only affected the polyphenols contents, but it also influenced the sugars concentration in cucumber leaves and specifically in cucumber fruits.
Significantly higher starch contents were found in cucumber leaves from high RH, while fructose, glucose and sucrose was not really effected by difference in RH. Stachyose and raffinose contents in leaves were significantly increased at moderate RH. On the other hand, fructose, glucose and starch was significantly higher in cucumber fruits from moderate RH.
Furthermore, total nitrogen, potassium and boron contents were higher in cucumber leaves from moderate RH. Higher total nitrogen, total carbon, phosphorous and potassium contents were found in cucumber fruits from moderate RH, while calcium, magnesium, manganese and molybdenum contents in fruits were increased with increased RH. The sensory evaluation of cucumber fruits indicated that RH induced a more bitter taste perception and contained more water. On the other hand, cucumber fruits from moderate RH was perceived more sweet, better in
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flavour and colour. Through correlation analysis, it was found that better flavour and good colour of cucumber fruits was due to starch contents and bitterness might be the taste of molybdenum.
In the second experiment, ABA application and MAP bags reduced the respiration of commercially produced cucumber fruits. Furthermore, the combined treatment of ABA with MAP bags effectively reduced physical weight loss, disease incidence and skin shrivelling of cucumber fruits. The ABA treated-MAP bagged and non ABA treated-MAP bagged fruits showed the lowest percentage of ion leakage, higher anti-oxidant capacity and total phenolics contents.
In summary, the cucumber plants grown at moderate RH performed better in terms of vegetative growth and produced good internal and external fruit quality. On the other hand, the cucumber fruits got extended storage life and maintained the better quality under MAP bagged condition.
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1. Introduction
Cucumber (Cucumis sativus. L) belongs to family Cucurbitaceae and is native to the sub- tropical regions of South Asia (Miao et al., 2007). Cucumber is produced in open fields as well as in controlled conditions of greenhouses depending upon climate and geographical locations (Nonnecke, 1989). The plant of cucumber is a creeping vine and bears cylindrical, yellowish - dark green fruits which are not only used as culinary vegetable but are also used in medicine and cosmetics products (Sarhan and Ismael, 2004).
The world production of cucumber is 75 million tons, while Asia is the main cucumber producing continent with a share of 88% followed by Europe and America with 7.5% and 2.8%
share respectively. In Asia, China is the major producer of cucumber with 54 million tons (FAOSTAT, 2014a). Mexico, Spain and Netherland are the major exporter of cucumber, while USA, Germany and Russia are the top three importers of cucumber (Anonymous, 2014). Due to unfavourable weather conditions in some countries such as Norway, China, Korea, Japan, Sweden, Netherland and Canada most of the cucumber are being produced under controlled conditions (Dorais et al., 2001)
Cucumber are consumed in different ways. It is consumed as fresh, raw, sauces, salad, pickle and as a component in many culinary dishes. Botanically cucumber is considered as fruit, while some consumers consider it as a vegetable (Malik and Bashir, 1994).
Cucumber contain 95.2% water. While, it is rich source of vitamin K (16%), antioxidants, phenols, potassium and some sugars (Bourn and Prescott, 2002).
It has been observed that the production of cucumber fruit at high RH in greenhouse reduces fruit quality in terms of nutritional value and shelf life, which is a serious problem (Beuchat, 1998). Cucumber fruits with a good quality at a moderate price is the demand of the market (Ahmed et al., 2004). Low shelf life due to loss of quality (membrane integrity, chilling injury, loss of chlorophyll, fruit weight loss and fruit softness) of many fruits in refrigeration is also a major concern of the consumers (Gine-Bordonaba et al., 2016). Cucumber is a tropical fruit and shows chilling injury (CI) symptoms at non-freezing temperature storage (Mao et al., 2007). Pre-harvest climatic conditions (light, relative air humidity and temperature) and postharvest storage temperature and packaging material are the key factors associated with cucumber fruit quality. Intensive production techniques and favourable environmental conditions
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in the greenhouse, give high yield and excellent quality as compared to cultivation in open fields (Bot, 2003). However, the climate during cucumber development is important for the quality.
Mostly, in greenhouse conditions cucumber production takes place at high relative air humidity (85-90%). To ventilate warm humid air out of the greenhouse is expensive when the temperature outside is low and many growers keep the greenhouse more or less closed to save energy during autumn/winter. Under these conditions, although producers get higher yield, bigger fruits, higher plant, and high leaf area index and thick leaf (Jeon et al., 2006), these conditions are not the desirable traits for commercial cucumber production. Moreover, due to high RH stomata functionality may be reduced and do not close in response to closure signals like darkness but continue to transpire also during night (Fordham et al., 2001). After harvest, cucumber fruit may also transpire, due to open stomata and loose fresh weight, physical quality and shelf life.
After harvest the quality of fruit can be maintained by adopting some protocols such as postharvest packaging material and storage temperature. Packaging material not only enhance the aesthetic value, but also influence the fruit physiology. Packaging material slows down the respiration rate of fruit by creating a micro-climate of low oxygen and higher carbon dioxide concentration, which reduces water loss, increases the storage duration and sustain the quality of the fruit.
1.1. Objectives of the Study
This thesis consists of two parts with different experimental approach: (1) Growth experiment in controlled environment with RH as the main factor to study quality of cucumber fruits (2) Postharvest storage experiment with cucumbers fruits from a commercial grower. In both parts, the objective was to attain the goal of premium fruit quality. The aim in the two different experiments was
To study how different relative air humility conditions during plant production and fruit development influence on plant growth and fruit quality.
To study the impact of different packaging materials on the storability of commercial fruits and to test if the plant hormone abscisic acid is important for the fruits sensitivity to cold storage.
5 1.2. Literature review
1.2.1. Cucumber
Cucumber is a widely grown creeping vine in the gourd family, which bears cucumiform shaped fruits that are usually used as a vegetable (Garden, 1893; Reznicek et al., 2011). It is divided into three main varieties: slicing, pickling, and seedless. There are several cultivars which have been created by using these main varieties. The cucumber is being produced all over the world (Grubben, 2004; Reznicek et al., 2011).
Cucumber grows up trellises or other supporting frames. Cucumber plant have Viburnum leaves with large leaf area (10-16cm), dark green in colour, cordate, apically acute and rough surface (Zomlefer, 1994; Grubben, 2004 and Reznicek et al., 2011). The fruit of typical cultivars of cucumber is indehiscent cylindrical, but glabrous, elongated with tapered ends with number of seeds. Cucumbers have smooth or warty surface, green to yellow in colour, weigh 50g to 4kg.
Each plant yields typically 25 fruits in a season (Grubben, 2004 and Lu et al., 2011).
1.2.2. Cucumber Industry
Cucumber is highly important commercial vegetable crop in the world. It ranks 4th following the potato, tomato and onion with an annual production of 75 million tonnes (FAOSTAT, 2014a). In Asia, China is major producer of cucumber with share of 57 million tonnes of total world’s production in 2014 (FAOSTAT, 2014a). After Asia, Europe is second largest producer of cucumber (Figure 1.1).
Figure 1.1: Worldwide Cucumber production by continents (FAOSTAT, 2014a). Source;
http://www.fao.org/faostat/en/#data/QC/visualize
6 1.2.3. Cucumber Industry in Norway
The agriculture industry in Norway shares 1.6% of total GDP (Gross domestic Product) (SSB, 2015). Cucumbers dominates other greenhouse crops and accounted for 51% of the total production. (SSB, 2015). While in overall vegetables commodities, cucumber ranks 3rd with respect to production (Figure 1.2).
Figure 1.2: Top four vegetable crops of Norway by annual production (FAOSTAT, 2014b).
Source; http://www.fao.org/faostat/en/#data/QC 1.2.4. Quality
In reported literature, there are a number of definitions of term “Quality” exist. But, according to Shewfelt (1999) “Quality is a term frequently used but rarely defined.” It means the definition of quality depends on stakeholders of supply chain groups i.e., producers, wholesalers, retailers and consumers. Usually, it is described in terms of physical appearance, biochemical compounds and sensory attributes (Cuartero and Fern´andez-Munoz, 1999). The general quality parameters of cucumber fruits are colour, size, surface smoothness, disease, fruit softness, skin bruises, shrivelling, physical injury, shelf life, taste, flavour and water contents (Ennis and O'sullivan, 1979). The cucumber fruit with more green in colour, big in size, have smooth surface, blemishes and disease free, good in taste and flavour are considered high in quality.
1.2.5. Nutritional Importance
Cucumbers are consumed fresh (Slicing cucumbers) and pickled. The juice from the cucumber leaves aid digestion and induce vomiting (Fern, 1997; Grubben, 2004). Most of the cucumber fruit biomass consist of 90-95% water. 100g serving of cucumber contains 3.63g of
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carbohydrates, 0.65g protein, 5% Pantothenic acid, 3% of Pyridoxine and Riboflavin, 147mg Potassium, 0.28mg Iron, 24mg Phosphorus and 13mg Magnesium. Surprisingly, they are a rich source of vitamin K and vitamin C with a share of 13.6% and 4.5% of dry matter respectively (Lixandru, 2014).
1.2.6. Therapeutic importance
In past few decades, the concept of using natural foods has changed and offered an advance practical approach through which consumers can attain optimal health by reducing the risk of chronic diseases (Bordbar et al., 2011). Cucumbers are rich source of vitamins (Vitamin K and C), phytochemicals (apigenin, quercetin and luteolin) and pinoresinol. Phytochemicals and pinoresinol have a strong antioxidant and anti-inflammatory effects (Anonymous, 2017).
Cucumbers also contains cucurbitacin compound, which is a phytonutrient and belongs to a large family of triterpenes. Cucurbitacin and pinoresinol has been reported to have an anti-cancer benefits (Lixandru, 2014).
Apart from antioxidant and anti-inflammatory activities of phenols in cucumber, the potassium in cucumber is also an important intracellular electrolyte. This ability of potassium intake reduces the blood pressure, control heart rate and minimize the chances of heart attack (Lixandru, 2014). The vitamin K in cucumbers plays a key role in promoting blood coagulation, osteoblastic (bone formation) activity and bone strengthening (Anonymous, 2017).
1.2.7. Climate and plant growth
Climatic factors are referred to abiotic factors and include water, rainfall, light, temperature, relative air humidity, CO2 and air movements. They influence plant growth and development directly and indirectly. A lot of research work has been reported on the effects of rainfall and water (Edmond et al., 1975; Eagleman, 1985; Miller, 2001), light (Devlin, 1975;
Edmond et al., 1975; Manaker, 1981; Abellanosa and Pava, 1987), temperature (Devlin, 1975;
Poincelot, 1980) and wind on plant growth and development. But, very little consideration have been given to influence of air movement and relative air humidity (Miller, 2001). All climatic factors are associated with photosynthesis, transpiration, transportation of water, plant growth and development and other physiological processes in plants.
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The fundamental process for carbon (C) accumulation, growth, and biomass production in plants is photosynthesis. All climatic factors such as quality and intensity of light, temperature and relative air humidity influence the photosynthesis. These factors also indirectly affect biomass production and plant growth (Bakker, 1995). Phytochrome, light receptor respond to light quality and triggers multi-component signals to induce fundamental cellular processes and controls the plant height (Reed et al., 1993). Low temperature reduces the water absorption and slows down the physiological process, which reduce the plant growth (Skálová et al., 2015).
These factors not only affect the plant growth, but also control external quality, internal quality and organoleptic attributes of vegetable products (Gruda, 2005).
1.2.8. Relative Air Humidity and Plant Morphology
High relative air humidity (RH) severely affects the plant production in greenhouses.
Most specifically, winter climate of Northern countries, is not quite friendly for ventilation and energy saving (prone to heat loss) (Mortensen, 2000). It has been reported that high RH have strong effects on plant morphology such as increase in plant height (Hoffman and Rawlins, 1971;
Mortensen, 1986; Mortensen and Gislerød, 1990; Mortensen and Fjeld, 1998; Mortensen, 2000;
Leuschner, 2002; Jeon et al., 2006), biomass and length of shoots (Hoffman et al., 1971;
Mortensen, 1986; Mortensen and Gislerød, 1990; Mortensen and Fjeld, 1998; Mortensen and Gislerød, 1999; Mortensen, 2000; Jeon et al., 2006), more leaf area (Mortensen, 2000; Leuschner, 2002; Jeon et al., 2006; Hovenden et al., 2012), less leaf thickness (Leuschner, 2002; Torre et al., 2003) and chlorophyll contents (Mortensen and Gislerød, 1990; Jeon et al., 2006).
The increase in leaf area index (LAI) has been linked to photosynthesis, carbon assimilation and carbon metabolism (Jeon et al., 2006). Torre et al. (2003) reported reduction in leaf thickness, which was attributed to a decrease in size of spongy and mesophyll cells under the epidermis of leaves. It was also observed that the size of intercellular air-spaces increased under high RH condition. Same kind of findings were showed by Leuscher (2002). Most of previous studies reported the increase in leaf area at high RH (Van de Sanden & Veen, 1992; Roriz et al., 2014), which is due to cell expansion in epidermal cells (Carins-Murphy et al., 2014). But Innes, (2015) stated that the leaf area and leaf length not really get influenced by RH level. Van de Sanden (1985) and a recent study by Jakobsen (2016) reported opposite findings, they found the increase in leaf area of cucumber plant at moderate RH.
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There are very few studies have reported the effects of RH on chlorophyll contents. Only one study stated the increase of relative chlorophyll contents in response to increase in RH (Jeon et al., 2006). On the other hand, Innes, (2015) and Jakobsen, (2016) found less chlorophyll contents in plants under high RH.
1.2.9. Carbohydrates and polyphenols in cucumber
Stachyose, raffinose, sucrose, glucose, fructose and starch were found in cucumber plant leaves, as mentioned before (Alam, 2016). While fructose, glucose, stachyose and starch are primary sugars of cucumber fruits. Most of previous studies reported effect of temperature and light on photosynthesis and carbohydrates production in plants (Taji et al., 2002). Riesmeier et al.
(1994) reported that these assimilates translocate in the form of sucrose, while in cucumber they also found as stachyose. That sugar is considered as predominant form of transport sugar in cucumber family (Hendrix, 1982; Webb and Gorham, 1964), but in fruits there was not stachyose found. On transportation to fruits, the stachyose metabolized into sucrose and later converted into glucose and fructose (Gross and Pharr, 1982). More photosynthesis take place at high RH, because stomata remain open and excessive CO2 remain available to the plant (Grange and Hand, 1987). Previously very little study have been done on photosynthesis activity and RH.
A lot of studies have been conducted regarding influence of RH on plant growth, transpiration, stomatal conductance, photosynthesis, transport of mineral and water (Hoffman and Rawlins, 1971; Ford and Thorne. 1974; Tibbitts and Bottenberg, 1976; Tibbitts, 1979; Gislerod, et al., 1987; Gislerod and Nelson, 1989; Gislerod and Mortensen. 1990; Bakker, 1991; Torre. et al., 2001; Carins- Murphy, et al., 2014). But very few studies have focused on the effect of RH on polyphenols in leaves or fruits. Cucumber plant contain a lot of polyphenols and some of them are identified as apigenin, quercetin, luteolin (Hertog, et al., 1992; Chu, et al., 2000; Lugast and Hovari, 2000) and pinoresinol (Peñalvo, et al., 2005; Milder, et al., 2005; Peñalvo, 2007). But little researched base information is available about RH effect on phenols concentration in plants.
1.2.10. Greenhouse relative humidity and Fruit quality
Preharvest relative air humidity in greenhouse not only influence the plant growth and physiology, but also effect the fruit quality and shelf life. High and low vapour pressure deficit (VPD) have various impacts on postharvest fruit quality of cucumber and tomatoes. Fruits, cut
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flowers, and ornamentals grown at high RH showed poor postharvest keeping quality, due to water loss, chilling injury at low temperature and less tolerance to stress (Mortensen and Fjeld, 1998; Mortensen, 2000; Torre et al., 2003). High RH (Low VPD) caused a decrease in the fresh weight of marketable tomatoes (Holder and Cockshull, 1990). Tomatoes grown at high RH face physiological and ripening disorders after harvest (Mulholland et al., 2001). Fricke and Krug (1997) reported a finding that the cucumber fruit lost the quality under various humidified treatments. Bakker et al. (1987) recommended that variation in day and night-time humidity provided excellent cucumber fruit quality and better storage life. Cucumber fruits contain stomata on peel and stomata of cucumber fruits grown at high RH behave same as stomata of plant leaves (Mortensen, 2000).
1.2.11. MAP bags and fruit physiology
The respiration is a metabolic process, which play major role in deterioration of the fresh produce, and it aims at the oxidative breakdown of complex organic substrates into simple molecules such as CO2 and H2O with the production of energy (Fonseca et al., 2002). Respiration rate of fresh produce depends on the storage conditions, particularly temperature, RH and gaseous composition. Respiration rate can be slow down by decreasing the O2 concentration as well as increasing the CO2 concentration in the environment (Saltveit, 2002; Rocculi et al., 2006).
Modified atmosphere packaging (MAP) is one of the most important food preservation methods.
By using MAP bags, the respiration rate slows down through creating the microclimate in the bags, which helps to extend the storage life and maintain the natural quality of fresh produce (Martínez-Ferrer et al., 2002).
1.2.12. MAP bags and chilling injury
Chilling injury (CI) is a common physiological disorder of many tropical and subtropical fruits, vegetables and ornamental crops which arises during the low temperature storage (Cabrera and Saltveit, 1990). Exposure of chilling-sensitive crops to cold temperature (<12 °C) caused variable symptoms that included uneven ripening or discoloration, higher water loss, increased surface pitting, wilting, fruit softening upon warming and increased permeability of the cellular membranes (Cabrera and Saltveit, 1990; Wang, 1993; Lelièvre et al., 1995). CI becomes more
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severe with longer storage times and/or at lower temperatures (Zagory and Kader, 1988).
Differences in chilling sensitivity have been reported in tomatoes (Autio and Bramlage, 1986).
Normally due to aminocyclopropane-1-carboxylic acid (ACC) synthase activity, the ethylene production is a common symptom of chilling injury (Wang, 1987). The production of polyamine titres increased in plants with CI and other stresses such as osmotic shock, variation in UV radiation, oxygen deficiency stress, low pH, as well as K+ and Mg2+ deficiency (Serrano, et al., 1997; Wang, 1987). Apart from ethylene production, the electrolyte or ion leakage is also associated with CI, fruits or some plant cells lost their membrane permeability at chilling temperature (McCollum and McDonald, 1991). In tomato fruits the ion leakage due to chilling injury did not show immediate increase on exposure to chilling temperature (Saltveit, 2002).
Treating the lemon and avocado fruits with CO2 (10−40%) before low temperature storage reduced CI symptoms, while avocado tolerated low temperature with treatment of low concentrations of O2 (Wang, 1987; Pesis, et al., 2000). On the other hand, modified atmospheres packaging diminish the chilling injury symptoms in many fruits (Cabrera and Saltveit, 1990). It has been reported that the Xtend® film (XF) was more effective to reduce the symptoms of CI in mango fruits as compared to micro-perforated polyethylene (PE) film (Pesis, et al., 2000).
1.2.13. Exogenous ABA application and fruit quality
Although plants produce ABA endogenously, but the exogenous application of ABA also influence the plant physiology and has been implicated as a regulatory factor (Heino, et al., 1999). Spomer (1979) reported that the exogenous application of ABA on cucumber seedlings increased the membrane integrity and reduced the chilling injury and ion leakage, while Rinkin et al. (1976) noticed that exogenous application of ABA increased the level of endogenous ABA and also increased the tolerance of plant tissues to chilling. Phenol concentration and phenylalanine ammonia-lyase (PAL) activity increased rapidly with exogenous application of ABA in strawberries fruits (Jiang and Joyce, 2003). Previously it has been reported that the opening of stomata regulated the elevation of Ca2+ in guard cells and down-regulated the K+ ions (Schroeder and Hagiwara, 1989) and H+-ATPases (Kinoshita et al., 1995), which provided basic and mechanized approach for ABA and influence of Ca2+ to inhibit K+ uptake.
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2. Materials and Methods
The present study was conducted at the Centre of climate regulated Plant Research (SKP), Norwegian University of life Sciences, Norway during May, 2016 to October, 2017. The experiments were conducted in 2 phases.
2.1. Experiment 1: effect of relative air humidity on plant growth and fruit quality 2.1.1. Seedling Production
The seeds of cucumber (Cocktail, Quarto F1 cultivar, L.O.G. As) were sown directly into 30 (12 cm) pots in a greenhouse compartment (20 – 25 °C, RH 70%, ambient CO2 and 100µmole.m-2.s-1 PAR from high pressure Sodium (HPS) lamps) on 9th May, 2016. The Sphagnum peat (pH 5.0 – 6.0 and salinity ca. 1.5 – 2.5) produced by Degernes Torvstrøfabbrikk AS, (Degernes, Norway) was used as growing media.
The climate in greenhouse was controlled by a PRIVA system (Priva, De Lier, The Netherlands). HPS lamps (Osram NAV T-400W, Munich, Germany) were used to meet the daily light requirement of plants and light intensity was measured with Li-Cor Model L1 250 Quantum Sensor (Li-Cor Inc., Lincoln, NE, USA). To attain the uniform germination, pots were covered with polyethylene sheet. On emergence of the seedling the sheet was removed. Seedlings were irrigated daily. On second leaves stage (27th May, 2016), the plants were moved to controlled environment growth chambers (Figure 2.1).
Figure 2.1: Cucumber Plants seedlings, before transfer to growth chambers
13 2.1.2. Experiment set-up
The 22 healthy plants were subjected to controlled relative air humidity growth chambers. 11 plants were placed in each chamber of moderate 60% and high 90 % RH (Figure 2.2). Other climatic conditions (23 °C temperature, ambient (400ppm) CO2 and 200µmol.m-2.s-1 PAR HPS lights with 20 light and 4 dark interval) were common in both chambers.
Figure 2.2: cucumber plants in growth chambers
2.1.3 Irrigation and plant maintenance
The plants were irrigated thrice a week with 50/50 mixture of KristalonTM Indigo (7.5%
NO3, 1% NH4, 4.9% P, 24.7% K, 5.7% S, 4.2% Mg, 0.027% B, 0.2% Fe, 0.06% Mn, 0.027% Zn, 0.004% Cu and 0.004% Mo, Yara Norge AS, Oslo, Norway) and YaraLiva® CalcinitTM Calcium nitrate solution (14.4% NO3, 1.1% NH4, 19.0% Ca, Yara Norge AS, Oslo, Norway) and 4 time a week with normal tap water.
The tendrils of plants were removed twice a week and on fruiting stage the pinching of diseased and small alternate fruits was also practiced twice a week. After one and half week (6th June, 2016) in growth chambers, 6 plants from each chamber were removed and used for initial data collection of growth parameters. Remaining 5 plants were left for fruiting. On fruit setting, 1, 2, 4 and 6 fruits were marked for fruit harvesting. The size of marked fruits were analyzed on alternate day to see maturation stage. On consistent reading, the fruits were considered ready to
14
harvest. The fruits harvesting was done on 29th June, 2016. 1 fruit from each plant was sampled for phytochemicals and sugars analysis. 1 fruit from each plant was used for dry matter percentage, minerals analysis, total phenols and antioxidants analysis, while remaining 2 fruits were used for sensory evaluation.
The data collection included physical parameter of plant and fruit growth, biochemical analysis and organoleptic evaluation. Furthermore fruit weight loss was also recorded. Analysis of dried fruits and leaves was also carried out to determine mineral concentration.
2.1.4 Data collection
2.1.5 Growth data and Physical Analysis
Following parameters were included in physical analysis.
Plant Height (measuring tape)
Number of Leaves (count)
Number of fruits (count)
Number of side shoots (count)
Average Leaf Area Index
Chlorophyll contents
Fruits Length (Measuring tape)
Fruit Diameter (Vernier Calliper)
Dry matter percentage
Every leaf from each plant was removed, counted, weighed (fresh sample weight) and used for measuring the leaf area (LA). The LA was measured by using LI-3100 Area meter (Li- Cor, Inc., Lincoln, Nebraska, USA). The leaf area was divided by counted leaves and average leaf area was calculated.
Chlorophyll contents were measured using CL-01 Chlorophyll content meter (Hansatech Instruments Ltd, Narborough Road, Pentney, King’s Lynn, Norfolk, UK). This field-portable, hand-held device determined relative chlorophyll content using dual wavelength optical absorbance (620 and 940nm) measurements from leaf samples. Relative chlorophyll content was displayed in the range 0 – 2000 units. The CL-01 features simple 2 button operation device. The reading was taken by placing the plant leaf between optics. It is auto-calibrating device.
15
The fruit length was measured from stylar end to blossom end. While fruit diameter was measured from both sides and centre of fruit. Dry matter was calculated on the basis of initial weight (before storage) and final weight (at the end of storage period) according to following formula;
Dry matter (%) = Final weight × 100 Initial weight
The leaves and fruits were divided into 2 parts. Half of leaves and fruits were used for dry matter contents (later on dried samples were used for minerals analysis) and remaining half were freeze dried and used for biochemical analysis.
2.1.6 Biochemical analysis
Biochemical analysis was done for the following compounds 1. Total phenolic and Anti-oxidants capacity (FRAP) 2. Phenolic compounds
3. Photo assimilates (Sugars)
2.1.6.1 Total Phenolic and Anti-oxidants capacity (Fruits) Sample collection and preparation
For measuring the anti-oxidant activity and total phenolic concentration, a KONE-lab was used. The fresh cucumber fruits (10 fruits) from each treatment (2 treatments) were stored at -20 ºC after harvest. The frozen samples were placed at room temperature for melting. After melting the sample was homogenized using hand blender. 3g of homogenized sample was taken in 50 ml centrifuge tube. The 30 ml of acidified (10 mM HCl) methanol was added in tube. The sample was vortexed for 30s. After vortex, the samples were sonicated in water bath at 0 ºC for 15 minutes followed by centrifugation for 10 minutes at 4 ºC and 4000 rpm. Supernatant was poured into Eppendorf-tube and centrifuged again for 3 minutes at 4 ºC and 132000 rpm.
2.1.6.1.1Anti-Oxidant activity (FRAP- assay)
The Ferric reducing ability of plasma (FRAP) assay was used to measure the concentration of total anti-oxidants. The method was based on the colour changes appeared when the TPTZ-Fe3+ (2,4,6-tri-pyridyl-s-trizine) complex was reduced to the TPTZ-Fe2+ form in the
16
process of antioxidants. An intense blue colour with the absorption maximum at 593 nm developed. The samples were measured at 600nm. An aqueous solution of 500 µM FeSO4 × 7.H2O was used for calibration of the instrument.
The calculation of standard was done by using following equation FRAP value of sample (µM) = Abs (sample) × FRAP value of Std
Abs (Std)
2.1.6.1.2 Total phenolic contents (TPC) determination
The TPC of cucumber was determined using the Folin–Ciocalteu (FC) method as outlined by Ainsworth and Gillespie (2007) with some modifications. The extracted samples (100 µL) were mixed with FC reagent (200 µL) in a fresh eppendrof tube and vortexed with the help of vortex mixer (SLV-6, MyLabTM, Seoulin BioScience, Korea) thoroughly for a few seconds.
After adding 800 µL of 700 mM sodium carbonate (Na2CO3) were again vortexed for few seconds and incubated at room temperature for 2 h. TPC were determined at 765 nm. The TPC were expressed as mg GAE 100g-1 against the standard curve of gallic acid (Figure 2.3).
Figure 2.3 Standard curve of gallic acid for determination of TPC 2.1.6.2 Polyphenols compounds
Sample preparation
After harvest, the leaves, fruit were peeled with common home use potato peeler. The peel and pulp samples were placed into test tubes (50 mL) and immediately frozen using liquid
17
nitrogen. The samples were placed into -80 ºC freezer. Before extraction the samples were freeze dried and grinded.
Procedure
The polyphenols were analyzed by high-performance liquid chromatography (HPLC) (Agilent, Series 1100, Germany), consisting of a binary pump (G1312A), a thermostated auto-sampler (G1329A), a thermostated column oven (G1316A) and a diode array detector (G1315B). The phenolic metabolites were separated using a Zorbax SB-C18 (4.6 × 60 mm) HPLC column (Agilent Technologies, USA). The samples were re-dissolved in 600 µL methanol: water (1:1) and eluted (flow rate 2 ml min-1) using the methanol: water gradient (Julkunen-tiitto et al., 1996).
The auto injection volume was 20 µL, and all runs were performed at 30 ºC. The phenolic metabolites were identified by comparing their retention times and UV spectrum with those of standards.
Extraction of phenolic compounds
20 mg of plant material (grinded) was taken into an Eppendorf vial and 600 µL MeOH (methanol) was added into the vial and homogenized for 30 s. The vial was left in ice bath for 15 minutes followed by centrifugation for 3 minutes on high speed (18000 rpm). The supernatant was poured into marked reagent vial (6 – 10 ml). 600 µL MeOH was added to residue (the rest of plant material) and homogenized again for 30 s. after that material was centrifuged for 3 minutes on high speed and supernatant was poured into same reagent vial. The same procedure was repeated 3 times more and supernatant was collected into reagent vial. MeOH was evaporated from the collected extract using vacuum concentrator. The extract was stored in 4°C until analysis.
HPLC analysis
The dried extract was removed from freezer. 200 µl MeOH and 200 µl distilled water was added into dried extract. The ultrasound bath was used to dissolve the material. The material was poured into Eppendorf vial and centrifuged. The supernatant was poured into a HPLC vial and a lid was put on. The sample was ready for analysis. The standard curve for some compounds was obtained from reported study (Figure 2.4a and 2.4b) (da Graça Campos & Markham, 2007).
18
Figure 2.4a: The HPLC Chromatograph showing Standard curve for luteolin-7, 3’-di-O- glucuronide and resveratrol.
19
Figure 2.4b: The HPLC Chromatograph showing Standard curve for apigenin, Fisetin, quercetin-2-O-glucoside, apigenin-7-O- [rhamnosyl(1-2) glucoside], Kaempferol and quercetin.
20 2.1.6.3 Carbohydrates
The amount of carbohydrates (stachyose, raffinose, glucose, sucrose and fructose) in leaves and fruits of cucumber were analyzed by following Gross and Pharr, (1982). From leaves and fruits 250 mg of freeze dried samples (2 treatments and 5 replications) were taken in each test tube. The carbohydrates were extracted through heating the samples in 1.5 ml of 80% ethanol at 70 ºC for 30 minutes using ultrasonic bath. After heating, the samples were centrifuged at 15000 rpm for 3 minutes. Supernatant from each tube was collected in separate tube. The ethanol was removed from the supernatant at 60 ºC by using the vacuum desiccator (Eppendorf AG 22331, 8 Hamburg, Germany). After that, 1 ml water was added into dried extract and heated at 60 ºC for 30 minutes followed by centrifugation at 15000 rpm for 3 minutes. The supernatant was collected separately and filtered through a 0.45 µm GHP membrane filter (Millipore) before HPLC.
Separation of Carbohydrates
After extraction, the samples were analyzed through HPLC. Carbohydrates were separated on the basis of their adsorption characteristics and it was analyzed by passing the solution through a column (Agilent Hi-Plex Ca USP L19, 4.0 * 250 mm). The separation was achieved through refractive index detector. For the mobile phase, water was used as solvent and flow rate was 0.3 ml/min and the temperature of column was 80 ºC. 10 µl of extracted sample was injected by the HPLC. Eluted carbohydrates were identified and quantified on the basis of their retention time and area of standard sugars (Figure 2.5).
Figure 2.5: The HPLC Chromatograph showing Standard curve for sugars (0.1% of each Stachyose, Raffinose, Sucrose, Glucose and Fructose) according to their retention time
21 2.1.6.4 Mineral analysis
Element analysis (Mg, Ca, S, P, Fe, K, B, Mo, Mn and Na) were performed on leaves and fruits samples taken at the time of harvest by using Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). Total nitrogen content was measured by the use of the Dumas method (Bremmer and Mulvaney, 1982). Five replications were taken of each treatment. Following process was adopted to accomplish the analysis.
Sample collection
(i) leaves sample (ii) fruit sample Leaves sample preparation
Steps involved are as follows (i) Leaves collection (ii) Drying
(iii) Crushing/grinding
After harvesting mature leaves were selected. Samples were kept in brown envelops with tiny holes made with punch machine. Leaves was further dried in oven at 60 ºC for 48 hours. After that, grinding was carried out with electric grinder until powder form. Samples were stored in labelled plastic vials.
Fruits sample preparation (for dry matter contents) (i) Weighing
(ii) Chopping (iii) Hot air drying (iv) Grinding/Crushing
100 gm of fruit was weighed with the help of weighing balance then chopping was carried out on a chopping mate with a sharp knife to divide the cucumber into minute pieces.
After that drying was done using a hot air oven at 60ºC, and samples were weighed periodically after 24 hours until the weight turn out to be constant after (48 to 50 hours). The final weight was noted to calculate dry matter using the following formula.
Dry matter % = Final weight ×100 Initial weight
Process was terminated on grinding. All samples were leaded towards minerals analysis.
22 2.1.7 Organoleptic Evaluation
The fruits were evaluated at ripening for organoleptic acceptability on the basis of taste, pulp colour, and texture and over all liking using the 9 point hedonic scale described by Peryam and Pilgrim (1957). Ten judges were called in the panel for organoleptic evaluation of treatments.
Hedonic scale (Peryam and Pilgrim, 1957)
Product: ____________ Date: ______________
Name of Judge: _________________________ Signature: __________________
Instructions: (Please read the instructions carefully before filling the blanks) 1. This is an organoleptic evaluation form for different cucumber samples.
2. Please follow the numerical system for scoring the samples.
Dislike extremely………..1 Like slightly…………...6 Dislike very much……….2 Like moderately………7 Dislike moderately………3 Like very much………..8 Dislike slightly………...4 Like extremely………...9 Neither like nor dislike………5
Please do not disturb the sequence of the samples provided.
Please wash the tongue before testing next sample, with the water provided.
Sr.No. Taste Pulp Colour Texture Flavour bitterness Water cont. Over Liking 1
2 3 4 5 6 7 8 9 10
23 2.2. Experiment 2: storability of commercial fruits
This experiment was conducted to analyse the impact of MAP bag, exogenous application of ABA and different temperature conditions influenced the cucumber fruit quality, storage duration and chilling injury sensitivity.
2.2.1. Experimental Layout Plant material
The plant material was comprised of physiologically mature green cucumber fruits with equal size and weight of 150 to 200 grams. The fruits were sourced from commercial green house (Espedal Hansdelsgarteri AS, Lier, Buskerud, Norway) on 3rd March, 2017. The average greenhouse conditions during production were as follow (Table 2.1).
Table 2.1: experiments layout
Temperature (°C) RH (%) CO2 (ppm)
20-22 80-85 1100-1150
Fruit were harvested manually along with the pedicel to avoid sap injury. After harvesting fruit were packed in corrugated boxes and transported to cold storage facility near Centre of climate regulated Plant Research (SKP), Norwegian University of Plant Sciences, Norway, using private vehicle at 20°C. Upon arrival at SKP, the fruit were graded, grouped, sprayed (half of fruits) with ABA (500µM), dried, packed in Xtend® MA/MH (modified atmosphere/ modified humidity) bags (StePac L.A. Ltd. Tefen, Israel) and plastic folio wrapping, placed in boxes, taken fresh weight, and subjected to low temperature storage conditions (11°C ±1; 85% RH) for 14 and 21 days (Table 2.2).
After each removal from storage room, the fruits were used for data regarding weight loss, physical appearance, chilling injury or fungal rot and ion leakage. While, bagged fruits were analysed for gases. After taking data, the fruits were wrapped again in plastic folio, weighed and placed at ambient temperature of 22-25 °C for 2 days (concept of retail market).
After 2 days, the data about weight loss and physical appearance was noted. After taking data the fruits were wrapped and placed in normal fridge at 5-6 °C: 70-80% RH for 2 days (concept of consumers). Later on final data was noted and fruits were sampled for ion leakage, total anti-oxidant and total phenolic compounds.
24 Table 2.2: experiments layout
Main Factor Sub-factor No. of Removals Replications
Map Bag ABA Treated 2 2
Non ABA 2 2
Plastic Folio Film ABA Treated 2 2
Non ABA 2 2
2.2.2. Data Collection
2.2.2.1. Gases concentration
The respiration of bagged fruits was analysed by using CO2 analyser (Anagas CO2
Analyser). The concentration of CO2 was measured in percentage.
2.2.2.2. Physical data 2.2.2.2.1. Weight Loss
Fresh weight loss was calculated on the basis of initial weight (before storage) and final weight (at the end of storage period) according to following formula;
Weight Loss (%) = Initial weight - Final weight × 100 Initial weight
2.2.2.2.2. Skin shrivelling
Skin shrivelling of cucumber fruits was assessed by using scales used by Malik and Singh (2005). Skin shrivelling was recorded by using the scale as follows; 1: nill; 2: <10%
affected area; 3: 10-25% affected area; 4: 25-50% affected area; 5: >50% affected area.
2.2.2.3. Ion Leakage
Electrolyte leakage was determined on eight disks (4 mm × 1 cm) taken with a cork borer from skin tissue from the surface. Disks were immersed in 20 mL of 0.3 M mannitol in glass vials, which were agitated at 20 °C for 120 min. Ion leakage was measured as the amount of increased conductivity (μS cm-1) of the solution. After that, disks were boiled for 30 min and cooled to room temperature and the total conductivity was measured. Chilling- induced ion leakage was expressed as the percent of the total conductivity leaked per hour (Gonzalez-Aguilar et al., 2000).
2.2.2.4. Total Phenolic and Anti-oxidants (Fruits)
For determination of total phenolic and anti-oxidants compounds the same procedure was adopted (as mentioned in experiment 1).
25
3. Results
3.1. Experiment 1
3.1.1. Comparison of growth and morphology of cucumber plants and fruits produced in different RH conditions
3.1.1.1 Growth and Morphological parameters 3.1.1.1.1 15 days under controlled conditions
After 15 days under controlled conditions, the plant growth and morphology of
‘Quarto F1’ cocktail cucumber were slightly affected by RH (RH). No significant effects of RH on average leaf area, relative chlorophyll contents and number of fruits were found.
However, the average leaf area (5.4%) and total chlorophyll contents (7.7%) were higher in plants grown at moderate RH compared to high RH. While the number of leaves and shoot length were significantly higher in plants grown at high RH with 20.5% and 35.4% difference respectively compared to moderate RH. Additionally the plants grown at moderate RH had 31.25% more side shoots compared to plants produced in high RH (Table 3.1).
Table 3.1: Growth and morphological Parameters of ‘Quarto F1’ cocktail cucumber plants after 15 days under different RH conditions (60% RH and 90% RH)
Parameters 60% RH 90% RH
Mean Mean SE LSD
Avg. leaf area per leaf (cm2) 139.35a 132.15a 5.6397 NS
Relative Chlorophyll content 19.53a 18.12a 1.0356 NS
No. of Leaves 7.8b 9.4a 0.3162 S
No. of Side Shoots 4.2a 3.2b 0.4243 S
Shoot length (cm) 42.140b 57.060a 1.9499 S
No. of Fruits 8.8a 10.0a 1.1489 NS
Means in rows not sharing similar letters differ significantly at P≤0.05; NS = Non-significant;
S= Significant
3.1.1.1.2 On Harvesting
At the stage of harvest (33 days after start of treatments) the vegetative growth and fruit morphology were significantly affected by the different RH conditions (Table 3.2).
Average Leaf Area, relative chlorophyll contents, percentage dry matter (biomass) of leaves and fruits, number of side shoots and fruit length were significantly higher in plants grown at moderate RH (60%) compared to high RH (90%) (Table 3.2). An increase in average leaf area
26
(7.5%), relative chlorophyll contents (17.2%), percentage dry matter (2.7%), number of side shoots (17.1%) and a higher number of fruits (18.9%) were observed in plants grown at moderate RH compared to high RH. On the other hand, a higher number of leaves (9.6%), longer shoots (9.0%) and an increase in fruit diameter (41.7%) were noticed in plant grown at high RH than moderate RH (Figure 3). While number of fruits were non significantly different, but a slightly higher number of fruits were found in plants grown in high RH conditions (Table 3.2).
Table 3.2: Morphological Parameters of ‘Quarto F1’ cocktail cucumber plants and fruit at the time of harvest under different RH conditions (60% RH and 90% RH), n = 9
Parameters
60% RH 90% RH
Mean Mean SE LSD
Average Leaf Area per Leaf (cm2) 143.63a 133.60b 2.0324 S
Relative Chlorophyll Contents 20.530a 17.520b 0.7490 S
Dry Matter Leaf (%) 15.274a 14.872b 0.0592 S
No. of Leaves 33.2b 36.4a 0.4472 S
No. of Side Shoots 16.4a 14.0b 0.4000 S
Shoot length (cm) 177.12b 193.08a 5.0930 S
No. of Fruits 29.6a 31.4a 1.1489 NS
Fruits diameter (mm) 23.396b 33.152a 1.6574 S
Fruit length (cm) 8.42a 7.08b 0.4391 S
Means in same row not sharing similar letters differ significantly at P≤0.05; NS = Non- significant; S= Significant
27
Figure 3: Pictorial view of cucumber fruits from different RH conditions.
3.1.2 Biochemical analysis of cucumber leaves and fruits under different relative air humidity conditions
3.1.2.1 Biochemical analysis
3.1.2.1.1 Total phenolics and Anti-oxidant capacity (FRAP)
Although total phenolic contents and anti-oxidant capacity in fruits were significantly affected by RH (appendix 1 and 2), and the anti-oxidant capacity was almost double in cucumber fruits produced in moderate RH as compared to high RH. Furthermore, the total phenolic contents were also higher (24.8%) in fruits sample of moderate RH (Figure 3.1).
Figure 3.1: FRAP (µM/L) and Total phenolics contents (mg/L) in ‘Quarto F1’ cocktail cucumber fruits from different RH conditions (60% RH and 90%RH) Vertical bars represent SE± 5.5138 and 0.7215, the different letters on the bars express significant difference, n=9 3.1.2.1.2 Polyphenols in cucumber plant leaves, fruit pulp and peel
The High Pressure Liquid Chromatography (HPLC) chromatograms of the phenolic fractions in cucumber leaves, fruit peel and fruit pulp were analysed. Some of the peaks were identified according to saved standards of polyphenols in the system library. According to the retention time, almost 18 peaks were common in each leaf sample (Table 3.3) and 3 of them were identified as resveratrol, luteolin and apigenin by comparing their spectrums with standard spectrum (Figure 3.2, 3.3 and 3.4). Unidentified polyphenols in the different treatments were compared on the basis of peak area. While identified polyphenols were compared on the basis of their concentration, which was measured by comparing the standard compound against samples measurements.
In leaves, resveratrol and luteolin and 3 unidentified peaks were significantly different between the RH treatments. While apigenin was non significantly affected by RH (Appendix
28
3). Higher concentration of resveratrol, luteolin and apigenin was observed in leaf samples from moderate RH (60% RH) as compared to leaf sampled from higher RH (90% RH) (Figure 3.7) and 2 unidentified polyphenols were also significantly higher in leaves samples from moderate RH. The remaining 12 peaks were non significantly different for both treatments (Table 3.3). Peak with retention time 21.8 showed higher value for polyphenols concentration in leaves samples from higher RH (90% RH) (Table 3.3).
Figure 3.7: a) Apigenin (mg/g), (b) luteolin (mg/g) and (c) resveratrol (mg/g) in ‘Quarto F1’
cocktail cucumber leaves from different RH conditions (60% RH and 90%RH) Vertical bars represent SE± 0.0454, 0.0206 and 0.178, the different letters on the bars express significant difference n=9
On the other hand, almost 8 peaks of different polyphenols was observed in fruits samples from both experimental treatments. 6 peaks were identified polyphenols (apigenin, luteolin, quercetin 3 glycoside, quercetin, pinoresinol and resveratrol) on the basis of their spectrum match (Figure 3.2, 3.3, 3.4, 3.5, and 3.6) and 2 peaks were found as unknown compounds
29
Figure 3.2: Resveratrol HPLC spectrum, red line presents the standard compound, blue line is spectrum of resveratrol in samples
Figure 3.3: Luteolin HPLC spectrum, red line presents the standard compound, blue line is spectrum of luteolin in samples
30
Figure 3.4: Apigenin HPLC spectrum, red line presents the standard compound, blue line is spectrum of apigenin in
samples
Figure 3.5: Quercetin HPLC spectrum, red line presents the standard compound, blue line is spectrum of quercetin in samples
31
Figure 3.6: Pinoresinol HPLC spectrum, blue line is spectrum of pinoresinol in samples
32
Table 3.3: Common peaks with retention time found in ‘Quarto F1’ cocktail cucumber leaves from different RH conditions (60% RH and 90%RH), n = 9
Retention time
Peaks
60% RH 90% RH
Leaves Leaves SE LSD
11.5 P1 1818.9a 1122.5b 131.67 S
12.5 P2 141.04a 71.475b 7.5488 S
13.5 P3 32.515a 15.35b 4.3347 S
14.8 P4 1178a 940.3a 198.3 NS
15.5 P5 164.26a 122.86b 9.5299 S
15.8 P6 722.16a 683.13a 74.757 NS
16 P7 1275.5a 1411.5a 203.71 NS
16.8 P8 432.72a 304.88b 36.247 S
17.5 P9 37.553a 28.553a 7.1314 NS
17.8 P10 33.425a 31.815a 10.273 NS
18.7 P11 41.758a 53.218a 16.67 NS
20.3 P12 202.92a 192.99a 45.51 NS
20.9 P13 65.293a 61.828a 10.815 NS
21.5 P14 174.1a 217.7a 29.448 NS
21.8 P15 242.07b 368.1a 49.929 S
22.5 P16 24.69a 35.417a 6.4105 NS
46.5 P17 412.51a 46.14a 342.81 NS
46.8 P18 149.86a 41.597a 83.054 NS
Means in rows not sharing similar letters differ significantly at P≤0.05; NS = Non-significant; S= Significant
33
For the fruits, the analysis was done for pulp and peel separately. All the polyphenols showed statistically significant different results in response to RH (Appendix 4). A higher contents of apigenin was observed in peel and pulp of fruits from moderate RH as compared to peel and pulp of fruits from high RH (Figure 3.8a). The luteolin was identified in pulp of fruits from 60% RH followed by pulp of fruits from 90% RH, while no significant difference between the two RH appeared (Figure 3.8b). It was observed that quercetin-3-glucoside only existed in the pulp of cucumber fruits and quercetin only existed in the peel of cucumber fruits. The concentration of both quercetin-3-glucoside and quercetin were significantly higher in pulp and peel of fruits from 60% RH as compared to 90% RH (Figure 3.8c and 3.8d). The pinoresinol contents was higher in peel of cucumber fruits as compared to pulp.
Higher amount of pinoresinol was noticed in peel of 60% RH followed by peel of 90% RH, pulp of 60% RH and pulp of 90% RH (Figure 3.8e). Furthermore, the concentration of resveratrol was not significantly different in pulp and peel of fruits from moderate RH, but significantly different in pulp and peel of fruits from higher RH. A higher content of resveratrol was observed in pulp of fruits produced in 90% RH followed by pulp and peel from fruits produced in 60% RH and peel of fruits from 90% RH (Figure 3.8f). Two unknown compounds was also observed with different retention time. The first unknown compound was only found in peel of fruits, while the second unknown compound was noticed in pulp and peel of fruits. Unkown compound 1 was higher in peel of fruits from moderate RH as compared to high RH (Figure 3.8g). The unknown compound 2 was significantly higher in pulp as compared to peel, but no significant difference for both treatments, while higher contents was found in pulp of 60% RH (Figure 3.8h).
34
Figure 3.8: a) Apigenin (mg/g), (b) luteolin (mg/g), (c) quercetin 3 Glycoside (mg/g) and (d) quercetin (mg/g) in ‘Quarto F1’ cocktail cucumber fruit pulp and fruit peel from different RH conditions (60% RH and 90%RH) Vertical bars represent SE± 0.00623, 0.1213, 0.00747 and 0.0107, the different letters on the bars express significant difference, n=9
35
Figure 3.8: e) Pinoresinol (mg/g), (f) resveratrol (mg/g), (g) Unknown 1 (area) and (h) Unknown 2 (area) in ‘Quarto F1’ cocktail cucumber fruit pulp and fruit peel from different RH conditions (60% RH and 90%RH) Vertical bars represent SE± 0.00937, 0.0762, 28.923 and 50.645, the different letters on the bars express significant difference, n=9
3.1.2.1.3 Carbohydrates in cucumber plant leaves and fruits
Through HPLC analysis 6 different carbohydrates peaks in leaves samples, while 4 peaks in fruits samples were detected. On the basis of standard solution’s retention time 6 peaks from leaves samples were found as fructose, glucose, raffinose, starch, stachyose and sucrose and 4 peaks of fruits samples were identified as fructose, glucose, starch and stachyose (3.10a and 3.10b).
36 3.10a: HPLC spectrum of sugars peaks separation in leaves samples.
3.10b: HPLC spectrum of sugars peaks separation in fruits samples.
37 3.1.2.1.3.1 Carbohydrates in Leaves
Among the leaves samples from different RH conditions, fructose, glucose and sucrose contents were non significantly different, while starch, stachyose and raffinose contents were significantly affected (Appendix 5).
The concentration of fructose in leaves was the same in both treatments (Figure, 3.11a), while the concentration of glucose was 16.8% higher in leaves from high RH as compared to leaves from moderate RH (Figure, 3.11b). The sucrose concentration in leaves from 60%RH was 15.4% higher than the samples from 90% RH (Figure, 3.11c).
Moreover, the concentration of starch in the leaves grown at 90% RH was 67% higher than the leaves from 60% RH (Figure, 3.11d). Whereas, the stachyose and raffinose concentration in leaves from 60% RH was more than double as compared to leaves from 90%
RH (Figure 3.11e and 3.11f).
Figure 3.11: a) Fructose (mg/g), (b) Glucose (mg/g), (c) Sucrose (mg/g) and (d) Starch (mg/g) in ‘Quarto F1’ cocktail cucumber plant leaves from different RH conditions (60% RH and 90%RH) Vertical bars represent SE± 0.0342, 0.0429, 0.0586 and 0.1725, the different letters on the bars express significant difference, n=9
38
Figure 3.11: e) Stachyose (mg/g), and (f) Raffinose (mg/g) in ‘Quarto F1’ cocktail cucumber leaves from different RH conditions (60% RH and 90%RH) Vertical bars represent SE±
0.1456, and 0.05, the different letters on the bars express significant difference, n=9 3.1.2.1.3.2 Carbohydrates in Fruits
On the other hand, the contents of fructose, glucose and stachyose in fruits samples from different RH conditions were significantly different, while for starch highly significant difference was observed (Appendix 6).
The concentrations of fructose and glucose in fruits sample from 60% RH was 50.5%
and 35% higher as compared to samples from 90% RH respectively (Figure 3.12a and 3.12b).
High content of starch was found in cucumber fruit samples. However, the starch concentration in fruits sample from 60% RH was almost 3 times higher than the samples from 90% RH (Figure 3.12c). The concentration of stachyose showed an opposite trend as compared to the other carbohydrates. About 11% higher content of stachyose was found in the samples from 90% RH as compared to fruits samples from 60% RH (Figure 3.12d).
Fi gure 3.12: a) Fructose (mg/g) and (b) Glucose in ‘Quarto F1’ cocktail cucumber fruits from different RH conditions (60% RH and 90%RH) Vertical bars represent SE± 0.0437, and 0.1326, the different letters on the bars express significant difference, n=9
